Influence factors of metagenomic next-generation sequencing negative results in diagnosed patients with spinal infection.

The aim of this study was to evaluate the influence factors of metagenomic next-generation sequencing (mNGS) negative results in the diagnosed patients with spinal infection. mNGS test was applied in a cohort of 114 patients with suspected spinal infection, among which 56 patients had a final diagnosis of spinal infection. mNGS achieved a sensitivity of 75.0% (95% CI, 61.6% to 85.6%) and a specificity of 84.5% (95% CI, 72.6% to 92.7%), using histopathology and culture results as reference. Diagnosed patients with a negative culture result had lower white blood cell account, percentage of neutrophilic granulocyte, C-reactive protein (all P<0.05) and relatively higher rate of prior antimicrobial treatment history (P=0.059). However, diagnosed patients with a negative mNGS result did not have such difference with mNGS-positive patients, suggesting that mNGS was not strictly limited by the above indicators, which presented the advantages of this technique from another point of view.

Exosomes Derived from Caerulein-Stimulated Pancreatic Acinar Cells Mediate Peritoneal Macrophage M1 Polarization and Pyroptosis via an miR-24-3p/MARCH3/NLRP3 Axis in Acute Pancreatitis.

OBJECTIVES: Acute pancreatitis (AP) has a high incidence of hospitalizations, morbidity, and mortality worldwide. A growing number of studies on AP pathogenesis are based on caerulein-induced experimental model, which simulates human AP in vivo. It has been demonstrated that both pancreatic acinar cells and peritoneal macrophages are involved in pancreatic inflammation and damage. However, their connection has not been well understood. METHODS: A caerulein-induced AP model was established on the pancreatic acinar cell line AR42J. Rat macrophages were isolated from the peritoneal cavity. The effects of caerulein-induced pancreatic exosomes on the peritoneal macrophage and pancreas in vivo and in vitro were examined. The underlying molecular mechanism was investigated by exploring the regulatory role of downstream molecules. RESULTS: We found that exosomes derived from caerulein-treated AR42J cells induced rat peritoneal macrophage M1 polarization and pyroptosis. miR-24-3p was upregulated in caerulein-stimulated exosomes, whereas the miR-24-3p inhibitor counteracted the effect of pancreatic exosomes on peritoneal macrophage M1 polarization and pyroptosis. Furthermore, miR-24-3p inhibited March3 expression, whereas MARCH3 mediated NLRP3 ubiquitination in rat peritoneal macrophages, which, in turn, contributed to the apoptosis, reactive oxygen species production, and inflammation in AR42J cells. CONCLUSIONS: Exosomes derived from caerulein-stimulated pancreatic acinar cells mediate peritoneal macrophage M1 polarization and pyroptosis via an miR-24-3p/MARCH3/NLRP3 axis in AP.

Comparison of postoperative recovery of primary pterygium excision combined with either limbal stem cell transplantation or amniotic membrane transplantation: a randomized controlled trial-based meta-analysis.

OBJECTIVE: To compare the postoperative recovery of primary pterygium excision combined with either limbal stem cell transplantation (LSCT) or amniotic membrane transplantation (AMT). METHODS: All relevant studies on the primary pterygium excision combined with either LSCT or AMT conducted before August 2022 were extracted from PubMed, EMBASE, Web of Science, and Cochrane Library databases. The main outcomes compared were tear film stability at 1, 3, and 6 months after surgery, postoperative corneal epithelial healing time, recurrence rate, and complications. RESULTS: Sixteen randomized controlled trials (RCTs) with 1390 eye cases were included in this meta-analysis. We found that patients of the AMT group improved significantly in the results of the tear break-up time (BUT) and Schirmer I test at 1 month after surgery (BUT: MD=-0.37, 95% CI: -0.62, -0.12, P<0.05; Schirmer I test: MD=-0.32, 95% CI: -0.57, -0.07, P<0.05) compared with those of the LSCT group, suggesting that the early stage of tear film stability after primary pterygium excision combined with AMT was superior to the LSCT combination. However, according to the Schirmer I test result, the patients in the LSCT group showed increased tear production compared to the AMT group at 3 and 6 months after surgery (3 months: MD=0.36, 95% CI: 0.08, 0.64, P<0.05; 6 months: MD=0.33, 95% CI: 0.07, 0.60, P<0.05), suggesting that the LSCT combination was superior to the AMT combination in long-term postoperative tear film stability. As for postoperative corneal epithelial healing time, the LSCT group exhibited shorter time than the AMT group (MD=-1.17, 95% CI: -2.15, -0.19, P<0.05). Furthermore, the recurrence rate was lower in the LSCT group than in the AMT group (RR=0.42, 95% CI: 0.30, 0.59, P<0.05). Lastly, there was no statistical difference in BUT and complication rate at 3 and 6 months after surgery between the LSCT and AMT groups. CONCLUSIONS: Our analysis suggests that primary pterygium excision combined with LSCT may be a better choice compared to the combination with AMT in postoperative recovery.

Procyanidin B2 alleviates oxidative stress-induced nucleus pulposus cells apoptosis through upregulating Nrf2 via PI3K-Akt pathway.

Oxidative stress can lead to nucleus pulposus cell (NPC) apoptosis, which is considered to be one of the main contributors to intervertebral disc degeneration (IVDD). Procyanidin B2 is a natural antioxidant that protects against oxidative stress. However, whether procyanidin B2 protects NPCs from oxidative stress remains unknown. In this study, we demonstrated that procyanidin B2 could reduce tert-butyl hydroperoxide-induced reactive oxygen species in rat NPCs and attenuate rat NPC apoptosis. Further experiments revealed that procyanidin B2 upregulated the expression of both nuclear factor erythroid 2-related factor 2 (Nrf2) and phosphorylation of protein kinase B (Akt). We then used silencing of Nrf2 and LY294002 to silence Nrf2 expression and block the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, respectively, and found that the protective roles of procyanidin B2 in NPCs were inhibited. Therefore, we demonstrated that procyanidin B2 alleviated rat NPC apoptosis induced by oxidative stress by upregulating Nrf2 via activation of the PI3K/Akt signaling pathway. This study provides a potential therapeutic approach for procyanidin B2 in IVDD, which might help in the development of new drugs for IVDD treatment.

Scutellarin-mediated autophagy activates exosome release of rat nucleus pulposus cells by positively regulating Rab8a via the PI3K/PTEN/Akt pathway.

To provide a basis for promising exosome-based therapies against intervertebral disc degeneration (IDD), our present research aimed to identify a mechanism underlying the vesicle release from nucleus pulposus cells (NPCs). Scutellarin (SC) is a natural chemotherapeutic agent isolated from Erigeron breviscapus with a variety of biological activities. Here, we observed the significantly elevated autophagy levels in rat NPCs under the stimulation of SC, leading to a concomitant enhancement of intracellular vesicle release, which could be attributed to the inactivation of the phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/protein kinase B (Akt) pathway. To ensure that exosome release was driven by SC via the autophagic pathway, we implemented gain-of-function and loss-of-function studies by additionally using insulin-like growth factor-1 (IGF-1) and small-interfering RNA of autophagy-related gene 5 (ATG5), and the exosome secretion decreased in the case of attenuated autophagy. Evidently, the treatment with SC exerted the remarkable upregulation of Rab8a through the overexpression of ATG5. After the respective knockdown of ATG5 and Rab8a, the increased release of exosomes induced by SC was reversed, whereas the number of intracellular vesicles was restored. Overall, it can be concluded that SC contributes to the autophagy activation in NPCs by acting on the PI3K/PTEN/Akt pathway, which upregulates the expression of Rab8a and promotes the release of exosomes, inspiring novel therapeutic strategies in preventing IDD that might be fruitfully investigated.

Hesitate between confocal laser endomicroscopy and narrow-band imaging: how to choose a better method in the detection of focal precancerous state of gastric cancer.

BACKGROUND: With a high incidence globally, deaths form gastric cancer (GC) are not rare. Early diagnosis is crucial to ameliorate its prognosis. Confocal laser endomicroscopy (CLE) and narrow band imaging (NBI) have been extensively applied in gastroscopy, particularly when it comes to the detection and management of premalignant gastric lesion. Our meta-analysis intends to appraise the diagnostic capability and compare the efficacy of NBI and CLE for focal precancerous state of gastric cancer. METHODS: We performed a literature search up to November 5, 2020 in online databases and major conferences. Two investigators assessed the methodological bias by QUADAS-2, followed by sophisticated study selection and data exaction to make a comparison between sensitivity, specificity, positive and negative likelihood values, and diagnostic odds ratio. A symmetric summary receiver-operating curve (sROC) and its area under the curve (AUC) were used to estimate threshold effect. Additionally, we evaluated the publication bias by Deeks' asymmetry test. RESULTS AND CONCLUSIONS: Four studies involved 248 patients and 526 lesions. In analysis drawn from every lesion, the NBI's pooled sensitivity and specificity were 87% (95% CI: 0.80-0.92) and 85% (95% CI: 0.75-0.91), and those of CLE were 90% (95% CI: 0.85-0.91) and 87% (95% CI: 0.83-0.91). CLE illustrated that the pooled two were slightly higher than NBI when compared at the level of every lesion. The AUC for NBI and CLE was 0.92 (0.90-0.94) and 0.95 (0.92-0.96), and there might be a threshold effect, according to the shoulder-like distribution of scatter points in the sROC. We did not find obvious publication bias in our meta-analysis.

Excessive mechanical strain accelerates intervertebral disc degeneration by disrupting intrinsic circadian rhythm.

Night shift workers with disordered rhythmic mechanical loading are more prone to intervertebral disc degeneration (IDD). Our results showed that circadian rhythm (CR) was dampened in degenerated and aged NP cells. Long-term environmental CR disruption promoted IDD in rats. Excessive mechanical strain disrupted the CR and inhibited the expression of core clock proteins. The inhibitory effect of mechanical loading on the expression of extracellular matrix genes could be reversed by BMAL1 overexpression in NP cells. The Rho/ROCK pathway was demonstrated to mediate the effect of mechanical stimulation on CR. Prolonged mechanical loading for 12 months affected intrinsic CR genes and induced IDD in a model of upright posture in a normal environment. Unexpectedly, mechanical loading further accelerated the IDD in an Light-Dark (LD) cycle-disrupted environment. These results indicated that intrinsic CR disruption might be a mechanism involved in overloading-induced IDD and a potential drug target for night shift workers.

TNF-α-stimulated nucleus pulposus cells induce cell apoptosis through the release of exosomal miR-16 targeting IGF-1 and IGF-1R in rats.

BACKGROUND: Exosomes may contain excess cellular components released by cells in response to harmful external stimuli to maintain cellular homeostasis. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), can induce cell apoptosis, alter cellular component expression levels, and stimulate exosome release. In this study, we examined whether exosomes released from nucleus pulposus cells (NPCs) under inflammatory conditions could induce normal NP cell apoptosis in rats and its underlining mechanism. METHODS: Exosomes were isolated from TNF-α-treated NPCs and used to treat normal NPCs. The effects were assessed by flow cytometry and western blot analysis. Anti-apoptotic insulin-like growth factor-1 (IGF-1) expression in NPCs was assessed by western blot analysis. Given the exosomal miRNAs might be the key factors of exosomes, bioinformatics approaches and quantitative real-time polymerase chain reaction (qRT-PCR) were used to identify IGF-1-regulating micro RNAs (miRNAs), including miR-16. Luciferase reporter assay assessed miR-16 regulation of IGF-1 and IGF-1 receptor (IGF-1R). NPCs were transfected with miR-16 mimic, and exosomes were applied to normal NPCs. NPCs were pretreated with 10 ng/mL TNF-α, transfected with miR-16 inhibitors, and the exosomes were isolated. Cell and exosome miR-16 levels were detected by qRT-PCR. Western blot analysis determined IGF-1, IGF-1R, and apoptotic marker levels in exosome-treated NPCs. RESULTS: Exosomes from TNF-α-treated NPCs induced apoptosis in normal NPCs and repressed IGF-1 expression. Exosomal miR-16 regulated IGF-1 and induced NPC apoptosis. The dual-luciferase reporter assay revealed that miR-16 binds the 3' untranslated regions (3'-UTRs) of IGF-1 and IGF-1R. Exosomal miR-16 repressed IGF-1 and the IGF-1R/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway which therefore induced NPC apoptosis. Rescue experiments using miR-16 inhibitors further validated these findings. CONCLUSIONS: The inflammatory factor TNF-α stimulated exosome release from NPCs, which induced the apoptosis of normal NPCs through the actions of exosomal miR-16. Exosomal miR-16 directly repressed the anti-apoptotic IGF-1/IGF-1R pathway, increasing the apoptosis of NPCs.

Injectable Mussel-Inspired highly adhesive hydrogel with exosomes for endogenous cell recruitment and cartilage defect regeneration.

In the early stage of osteoarthritis (OA), cartilage degradation in the surface region leads to superficial cartilage defect. However, enhancing the regeneration of cartilage defect remains a great challenge for existing hydrogel technology because of the weak adhesion to wet tissue. In the present study, an injectable mussel-inspired highly adhesive hydrogel with exosomes was investigated for endogenous cell recruitment and cartilage defect regeneration. The hydrogel with high bonding strength to the wet surface was prepared using a crosslinked network of alginate-dopamine, chondroitin sulfate, and regenerated silk fibroin (AD/CS/RSF). Compared with commercial enbucrilate tissue adhesive, the AD/CS/RSF hydrogel provided a comparative lap shear strength of 120 kPa, with a similar gelation time and a higher capacity for maintaining adhesive strength. The AD/CS/RSF/EXO hydrogel with encapsulated exosomes recruited BMSCs migration and inflation, promoted BMSCs proliferation and differentiation. Most importantly, the AD/CS/RSF/EXO hydrogel accelerated cartilage defect regeneration in situ, and extracellular matrix remodeling after injection in rat patellar grooves. The exosomes released by the hydrogels could recruit BMSCs into the hydrogel and neo-cartilage via the chemokine signaling pathway. Our findings reveal an injectable and adhesive hydrogel for superficial cartilage regeneration, which is a promising approach for minimally treating cartilage defect with arthroscopic assistance.

Adaptation in the Dorsal Belt and Core Regions of the Auditory Cortex in the Awake Rat.

The rat auditory cortex is divided anatomically into several areas, but little is known about the functional differences in information processing among these areas. Three tonotopically organized core fields, namely, the primary (A1), anterior (AAF), and ventral (VAF) auditory fields, as well as one non-tonotopically organized belt field, the dorsal belt (DB), were identified based on their response properties. Compared to neurons in A1, AAF and VAF, units in the DB exhibited little or no response to pure tones but strong responses to white noise. The few DB neurons responded to pure tones with thresholds greater than 60 dB SPL, which was significantly higher than the thresholds of neurons in the core regions. In response to white noise, units in DB showed significantly longer latency and lower peak response, as well as longer response duration, than those in the core regions. Responses to repeated white noise were also examined. In contrast to neurons in A1, AAF and VAF, DB neurons could not follow repeated stimulation at a 300 ms inter-stimulus interval (ISI) and showed a significant steeper ISI tuning curve slope when the ISI was increased from 300 ms to 4.8 s. These results indicate that the DB processes auditory information on broader spectral and longer temporal scales than the core regions, reflecting a distinct role in the hierarchical cortical pathway.

Autophagy-activated nucleus pulposus cells deliver exosomal miR-27a to prevent extracellular matrix degradation by targeting MMP-13.

Although autophagy may be beneficial for maintaining the metabolic balance of the extracellular matrix (ECM) in the nucleus pulposus (NP) and its vitality under inflammation, the underlying mechanism still remains unclear. A previous study found that autophagy activation stimulated the release of exosomes in normal chondrocytes, which are located in a similar avascular environment and share many common features with those of nucleus pulposus cells (NPCs). This study explored the protective effect on matrix degradation in the NP by exosomes derived from autophagy-activated NPCs and exosomal microRNAs. NPCs-derived exosomes (NPCs-Exos) were isolated from culture medium of either normal NPCs or rapamycin-treated NPCs and quantified by nanoparticle tracking analysis. The effect of rapamycin-treated NPC-derived exosomes on NPCs were assessed by coculture with interleukin 1ß (IL-1ß)-stimulated NPCs. After examination of six major proteinases of the ECM, matrix metalloproteinase 13 (MMP-13) was chosen for further study. miR-27a, which targets MMP-13, was investigated through previous studies and bioinformatics tool. The levels of miR-27a were upregulated in both rapamycin-treated NPCs and their exosomes, compared to the control. When exosomal miR-27a was transferred into NPCs, it alleviated IL-1ß-induced degradation of the NPC ECM by targeting MMP-13. Autophagy activation may promote the release of NPCs-derived exosomes and thereby prevent the NPC matrix from degradation. Autophagy activation also alleviates intervertebral disc degeneration (IDD), at least partly via exosomal miR-27a, which restrains MMP-13 expression under IL-1ß stimulation. Our work elucidates a new mechanism for how autophagy may participate in preventing IDD, which may be a promising therapeutic strategy.

Autophagy regulates exosome secretion in rat nucleus pulposus cells via the RhoC/ROCK2 pathway.

Our present study investigated whether exosome secretion of nucleus pulposus cells (NPCs) is regulated by autophagy. Different autophagic states of NPCs were induced by rapamycin (Rap), bafilomycin A1 (Baf) and other agents, and it was found that exosomes were secreted in an autophagy-dependent manner. Activation or inhibition of autophagy increased or decreased, respectively, the amount of exosomes that were released into the extracellular space. In addition, in order to confirm that Rap-promoted release of exosomes was mediated by autophagy rather than other pathways, we used autophagy associated gene 5 (ATG5) small-interfering RNA (siRNA) to silence the expression of ATG5 gene, which is indispensable for autophagy. The results showed that siRNA against ATG5 (siATG5) induced an accumulation of intraluminal vesicles (ILVs) in NPCs and a concomitant decrease in the amount of exosomes isolated from supernatant. Ras homolog gene (Rho) and Rho-associated coiled-coil forming protein kinase (ROCK) family molecules are capable of cytoskeletal remodeling and affecting vesicle transport. Therefore, we carried out targeted interventions and evaluated the effects of the RhoC/ROCK2 pathway on the secretion of exosomes within autophagic environment. Knockdown of RhoC and ROCK2 with corresponding siRNA significantly inhibited the secretion of exosomes originating from ILVs in NPCs, even when NPCs were subsequently treated with Rap. Taken together, our findings suggest that autophagy positively regulates expression levels of RhoC and ROCK2, and that the RhoC/ROCK2 pathway exerts a key function on NPCs-derived exosome secretion.